Leo H. Buhler

Cloning and Functional Expression of the Human Islet GLP-1 Receptor: Demonstration That Exendin-4 Is an Agonist and Exendin-(9–39) an Antagonist of the Receptor

A complementary DNA for a glucagon-like peptide-1 receptor was isolated from a human pancreatics islet cDNA library. The isolated clone encoded a protein with 90% identity to the rat receptor. In stably transfected fibroblasts, the receptor bound [125I]GLP-1 with high affinity (Kd = 0.5 nM) and was coupled to adenylate cyclase as detected by a GLP-1-dependent increase in cAMP production (EC50 = 93 pM). Two peptides from the venom of the lizard Heloderma suspectum, exendin-4 and exendin-(9–39), displayed similar ligand binding affinities to the human GLP-1 receptor. Whereas exendin-4 acted as an agonist of the receptor, inducing cAMP formation, exendin-(9–39) was an antagonist of the receptor, inhibiting GLP-1–induced cAMP production. Because GLP-1 has been proposed as a potential agent for treatment of NIDDM, our present data will contribute to the characterization of the receptor binding site and the development of new agonists of this receptor.

Impact of recipient age on outcome of ABO-incompatible living-donor liver transplantation.

Background. Transplantation of hepatic grafts from ABO-incompatible donors is controversial because of the risk of hyperacute rejection mediated by preformed anti-ABO antibodies. The aim of the present study was to evaluate the outcome of liver transplants performed with ABO-incompatible living-donor livers and to detect risk factors for development of complications. Methods. From June 1990 to February 2000, 66 patients, 10 months to 55 years old (median, 2 years old), received 68 ABO-incompatible living-donor liver grafts. The antibody titer and clinical course were followed prospectively during a period ranging from 3 to 11 years. Results. The 5-year patient survival was 59%, 76%, and 80% for ABO-incompatible, ABO-compatible, and ABO-identical grafts, respectively (P <0.01). In patients <1 year old, ≥1 to <8, ≥8 to <16, and and ≥16 years old, 5-year survival was 76%, 68%, 53%, and 22%, respectively. The incidence of intrahepatic biliary complications and hepatic necrosis in ABO-incompatible living-related grafts (18% and 8%, respectively) was significantly (P <0.0001) greater than in ABO-compatible and ABO-identical grafts (both 0.6% and 0%, respectively). Predictive risk factors for increased mortality and morbidity were age greater than 1 year and elevated anti-ABO titers before transplantation. Conclusions. ABO-incompatible liver transplantation was carried out with relative safety in infants <1 year old but was not satisfactory in children >1 year in long-term follow-up. Patients aged >8 years remain at considerable risk of early fatal outcome because of hepatic necrosis, and new strategies to prevent antibody-mediated rejection are required.

Unsaturated fatty acids inhibit the expression of tumor suppressor phosphatase and tensin homolog (PTEN) via microRNA-21 up-regulation in hepatocytes†‡

Phosphatase and tensin homolog (PTEN) is a regulator of phosphoinositide 3‐kinase signaling and an important tumor suppressor mutated/deleted in human cancers. PTEN deletion in the liver leads to insulin resistance, steatosis, inflammation, and cancer. We recently demonstrated that unsaturated fatty acids trigger steatosis by down‐regulating PTEN expression in hepatocytes via activation of a mammalian target of rapamycin (mTOR)/nuclear factor kappa B (NF‐κB) complex, but the molecular mechanisms implicated in this process are still unknown. Here, we investigated potential genetic and epigenetic mechanisms activated by fatty acids leading to PTEN down‐regulation. Our results indicate that unsaturated fatty acids down‐regulate PTEN messenger RNA expression in hepatocytes through mechanisms unrelated to methylation of the PTEN promoter, histone deacetylase activities, or repression of the PTEN promoter activity. In contrast, unsaturated fatty acids up‐regulate the expression of microRNA‐21, which binds to PTEN messenger RNA 3′‐untranslated region and induces its degradation. The promoter activity of microRNA‐21 was increased by mTOR/NF‐κB activation. Consistent with these data, microRNA‐21 expression was increased in the livers of rats fed high‐fat diets and in human liver biopsies of obese patients having diminished PTEN expression and steatosis. Conclusion: Unsaturated fatty acids inhibit PTEN expression in hepatocytes by up‐regulating microRNA‐21 synthesis via an mTOR/NF‐κB–dependent mechanism. Aberrant up‐regulation of microRNA‐21 expression by excessive circulating levels of fatty acids exemplify a novel regulatory mechanism by which fatty acids affect PTEN expression and trigger liver disorders. (HEPATOLOGY 2009.)

Human islet transplantation: lessons from 13 autologous and 13 allogeneic transplantations.

BACKGROUND A series of 13 islet autotransplantations and 13 islet allotransplantations performed between 1992 and 1999 at the University Hospital of Geneva are presented. Factors affecting the outcome are analyzed. METHODS Islet autotransplantation has been performed in seven patients with chronic pancreatitis and in six patients with benign tumors undergoing extensive pancreatectomy. Islet allografts were performed in C-peptide-negative patients simultaneously or after a kidney or lung transplantation. Each recipient received islets from one to four donors. Panel-reactive antibodies were monitored by microlymphocytotoxicity test. RESULTS Eleven of 13 patients who underwent autotransplantation maintained insulin independence for 6 months to 5 years. Two years after autologous islet transplantation, five of nine patients were insulin independent with an glycosylated hemoglobin of 5.9%. Three late islet failures occurred in patients with chronic pancreatitis. Islet yield was significantly lower in patients with chronic pancreatitis than in patients with benign tumors (2044 equivalent islet number/gram resected pancreas versus 5184 equivalent islet number/gram; P=0.037). In islet allotransplantation, no early graft loss was found. All 13 patients who underwent allotransplantation had basal C-peptide levels above 0.3 nmol/L for 3 months to 5 years. Mean glycosylated hemoglobin decreased from 9.1% before transplantation to 5.5% at month 3. Insulin independence was achieved in two type I diabetic patients. In four of six patients with graft failure, the graft had induced panel-reactive antibodies. CONCLUSIONS In islet autotransplantation, the reduced number of islets that can be isolated from fibrotic pancreata may be the major limiting factor. In islet allotransplantation, early graft function can now be consistently achieved. Islet allografts seem to be highly immunogenic, and chronic islet failure cannot be prevented consistently by conventional immunosuppression.

Recent results of elective open cholecystectomy in a North American and a European center: Comparison of complications and risk factors

Results of elective open cholecystectomy in 1252 patients treated in a North American and a European center were examined using a recent standardized classification of complications. Although there were significant differences between centers in population age, rate of concomitant disorders, and numbers of operators, the frequency and severity of complications were comparable. There were no deaths, but 12% and 14% of the patients developed complications in the two centers. About 6% of the patients developed grade I complications. Grade II complications were noted in 6% and 8%, and grade III in 0% and 0.3%. Using univariate and multivariate analysis, individual risk factors for developing complications were found to be different in the two centers. Two preoperative scoring systems, ASA and a simplified APACHE II, were predictive for complications in both centers, but did not account for all risk in these patients. Data from the two centers could not be combined because of significant interaction between risk factors and center. Elective open cholecystectomy is a safe procedure, particularly in terms of highly morbid complications and death. Generalization of risk factors identified in a particular center may be misleading because local conditions may significantly affect risk factors for complications. The data also demonstrate the advantages of a uniform way of reporting surgical complications, which may permit meaningful comparisons among centers.

Polyfunctional HCV-specific T-cell responses are associated with effective control of HCV replication

HCV infection has a severe course of disease in HIV/HCV co‐infection and in liver transplant recipients. However, the mechanisms involved remain unclear. Here, we evaluated functional profiles of HCV‐specific T‐cell responses in 86 HCV mono‐infected patients, 48 HIV/HCV co‐infected patients and 42 liver transplant recipients. IFN‐γ and IL‐2 production and ability of CD4 and CD8 T cells to proliferate were assessed after stimulation with HCV‐derived peptides. We observed that HCV‐specific T‐cell responses were polyfunctional in HCV mono‐infected patients, with presence of proliferating single IL‐2‐, dual IL‐2/IFN‐γ and single IFN‐γ‐producing CD4+ and dual IL‐2/IFN‐γ and single IFN‐γ‐producing CD8+ cells. In contrast, HCV‐specific T‐cell responses had an effector profile in HIV/HCV co‐infected individuals and liver transplant recipients with absence of single IL‐2‐producing HCV‐specific CD4+ and dual IL‐2/IFN‐γ‐producing CD8+ T cells. In addition, HCV‐specific proliferation of CD4+ and CD8+ T cells was severely impaired in HIV/HCV co‐infected patients and liver transplant recipients. Importantly, “only effector” T‐cell responses were associated with significantly higher HCV viral load and more severe liver fibrosis scores. Therefore, the present results suggest that immune‐based mechanisms may contribute to explain the accelerated course of HCV infection in conditions of HIV‐1 co‐infection and liver transplantation.

Successful treatment of recurrent focal segmental glomerulosclerosis after kidney transplantation by plasmapheresis and rituximab

A 22‐year‐old patient whose primary kidney disease was focal segmental glomerulosclerosis (FSGS) developed severe recurrence of proteinuria (up to 57 g/24 h) immediately after a haploidentic living donor kidney transplantation despite pre‐operative plasmapheresis. The immunosuppressive treatment consisted of tacrolimus, mycophenolate mofetil, basiliximab and steroids. He underwent 10 plasmapheresis sessions in the first 3‐week post‐transplantation. In addition, he received 2 i.v. doses of rituximab (RTX) 600 mg (375 mg/m2) on days 7 and 15. Proteinuria decreased below nephrotic range at day 14 and serum creatinine returned progressively to normal values. A short course of oral ciclophosphamide (100 mg/j) was administrated between days 22 and 40 and three additional plasmapheresis sessions on days 34, 39 and 49. This strategy allowed obtaining sustained full remission of the nephrotic syndrome (NS) and excellent graft function, which persists over 2 years after transplantation. No notable adverse events related to RTX or plasmapheresis were observed. This case suggests that RTX associated with plasmapheresis may be an effective treatment of recurrent NS because of FSGS.

Cx36 makes channels coupling human pancreatic β - cells, and correlates with insulin expression

Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observed in type 2 diabetes. Since human islets differ in several respects from those of laboratory rodents, we have now screened human pancreas, and islets isolated thereof, for expression of a variety of connexin genes, tested whether the cognate proteins form functional channels for islet cell exchanges, and assessed whether this expression changes with beta-cell function in islets of control and type 2 diabetics. Here, we show that (i) different connexin isoforms are differentially distributed in the exocrine and endocrine parts of the human pancreas; (ii) human islets express at the transcript level different connexin isoforms; (iii) the membrane of beta-cells harbors detectable levels of gap junctions made of Cx36; (iv) this protein is concentrated in lipid raft domains of the beta-cell membrane where it forms gap junctions; (v) Cx36 channels allow for the preferential exchange of cationic molecules between human beta-cells; (vi) the levels of Cx36 mRNA correlated with the expression of the insulin gene in the islets of both control and type 2 diabetics. The data show that Cx36 is a native protein of human pancreatic islets, which mediates the coupling of the insulin-producing beta-cells, and contributes to control beta-cell function by modulating gene expression.

Transplantation of mesenchymal stem cells for the treatment of liver diseases, is there enough evidence?☆

Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been extensively investigated in small animal models to treat both acute and chronic liver injuries. Mechanisms of action are not clearly elucidated but may include their ability to differentiate into hepatocyte-like cells, to reduce inflammation, and to enhance tissue repair at the site of injury. This approach is controversial and evidence in large animals is missing. Side effects of MSC infusion such as the contribution to a fibrotic process have been reported in experimental settings. Nevertheless, MSCs moved quickly from bench to bedside and over 280 clinical trials are registered, of which 28 focus on the treatment of liver diseases. If no severe side-effects were observed so far, long-term benefits remain uncertain. More preclinical data regarding mechanisms of action, long term safety and efficacy are warranted before initiating large scale clinical application. The proposal of this review is to visit the current state of knowledge regarding mechanisms behind the therapeutic effects of MSCs in the treatment of experimental liver diseases, to address questions about efficacy and risk, and to discuss recent clinical advances involving MSC-based therapies.

Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver.

Multipotent mesenchymal stromal cells (MSC) are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments) when compared to adult MSC (2/10 experiments). However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age. These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.